1. Field of the Invention
This invention relates generally to the introduction into proteins of amino acid segments having a biological targeting function which directs the proteins to cellular vesicles where they are processed into peptide residues recognized by the major histocompatibility complex class II proteins and to the use of this procedure to enhance the immune response of mammals to these targeted proteins as antigens.
2. Review of Related Art
A. Antigen Processing and Presentation to T Cells PA1 B. Lysosomal/Endosomal Compartment PA1 C. Other Proteins Found in the Endosomal/Lysosomal Compartment PA1 D. Lysosomal/Endosomal Targeting Signals PA1 E. Vaccine Development PA1 F. Cancer Vaccines PA1 * EBV Epstein-Barr virus gene products in Hodgkin's lymphomas as well as Burkits and other lymphomas, products of the HTLV-1 genome in adult T cell leukemia and human papillomavirus (HPV) E6 and E7 gene products in cervical carcinoma. PA1 * Mutations in various oncogenes such as the position 12 mutation in K ras have been implicated as a major genetic alteration of colon cancer as well as other malignancies. PA1 * Mutations in tumor suppressor genes such as P53 are extremely common in many malignancies. PA1 * Rearrangements that result in activation of oncogenes such as the rearrangement between the BCR and abl gene in chronic myelogenous leukemia generate notel protein sequences. PA1 * Tumors re-express developmental or embryonic genes which are not expressed in normal cells in the individual. Such an example is the MAGE gene identified as a source of a T cell recognized antigen in human melanoma.
The generally held theory for the mechanism of antigen recognition and response in the mammalian immune system is that there are two parallel cellular systems of T cells and antigen presenting molecules which distinguish between two types of antigens, foreign antigens introduced from outside of the cell (such as foreign chemicals, bacteria, and toxins) and endogenous antigens produced within the cell (such as viruses or oncogene products). It is now clear that the cell type which distinguishes between antigen types in the cellular immune response is the T cell. Via its heterodimeric T cell receptor, the T cell recognizes peptide fragments of these antigens presented as a complex with major histocompatibility (MHC) molecules (Yewdell and Bennenk, Cell, 62:203, 1990; Davis and Bjorkman, Nature, 334:395, 1988).
There are two general classes of MHC molecules, MHC class I and MHC class II proteins. These MHC molecules bind to antigens and present them to one of the two types of the T cell class of white blood cells, cytotoxic T cells (T.sub.c) or helper T cells (T.sub.h). MHC class I molecules present peptide antigens generally derived from endogenously produced proteins to the CD8.sup.+ T.sub.c cells, the predominant cytotoxic T cell that is antigen specific. MHC class II molecules generally present antigens that are introduced from without the cells, utilizing a distinct pathway for antigen presentation that involves generation of peptide fragments in endosomal/lysosomal organelles. MHC class II molecules are also found in these acidic organelles, co-localized with the invariant chain, a membrane glycoprotein that binds MHC class II proteins in the endoplasmic reticulum and is replaced by the antigenic peptides. After binding of the antigen to the class II molecules, the antigen/MHC II complex is brought to the cell surface for antigen presentation to CD4.sup.+ T.sub.h cells.
The functions of the two types of T cells are significantly different, as implied by their names. Cytotoxic T cells act to eradicate intracellular pathogens and tumors by direct lysis of cells and by secreting cytokines such a .gamma.-interferon. Helper T cells can also lyse cells, but their primary function is to secrete cytokines that promote the activities of B cells and other T cells and thus they broadly enhance the immune response to foreign antigens, including antibody-mediated and T.sub.c -mediated response mechanisms.
CD4.sup.+ T cells are the major helper T cell phenotype in the immune response. Their predominant function is to generate cytokines which regulate essentially all other functions of the immune response. Animals depleted of CD4.sup.+ or humans depleted of CD4.sup.+ cells (as in patients with AIDS) fail to generate antibody responses, cytotoxic T cell responses, or delayed type hypersensitivity responses. These results suggest that helper T cells are critical in regulating immune responses.
CD4.sup.+ MHC class II restricted cells have also been shown to have cytotoxic capacity in a number of systems. One of the most important disease-relevant cases in which CD4.sup.+ cytotoxic T cells have been demonstrated is in the response to fragments of the HIV gp120 protein (Polydefkis, et al., J. Exp. Med., 171:875, 1990). CD4.sup.+ MHC class II restricted cells have also been shown to be critical in generating systemic immune responses against tumors. In an adoptive transfer model, CD4.sup.+ cells are critical in eliminating FBL tumors in mice. In the active immunotherapy model of Golumbek, et al. (1991, Science, 254:713), CD4.sup.+ cells have also been shown to be critical in the systemic immune response against a number of different solid malignancies.
For all these reasons there has been increased interest in developing strategies that will most effectively activate MHC class II restricted CD4.sup.+ cells against a given specific antigen. Furthermore, CD4.sup.+ MHC class II restricted cells appear to be the critical memory cells in the T cell arm of the immune response. Therefore, an appropriate vaccination strategy is to generate CD4.sup.+ antigen-specific MHC class II-restricted memory T cell populations.
In keeping with the different functions of the cytolytic T cells and helper T cells, the tissue distribution of the MHC molecules that present antigens to these cells is markedly different. The MHC I protein complex that recognizes self or viral antigens is found in virtually all cell types, whereas the MHC II protein that reacts with foreign antigens is found largely in immune cells such as macrophages and macrophage-like cells that either secrete cytokines necessary for T.sub.h cell stimulation of B cells or that require the T.sub.h cell cytokines for their own stimulation. Cells exhibiting MHC II protein are generally called antigen presenting cells.
The processing and presentation of self or foreign antigens to the MHC I or MHC II complex, respectively, generally occurs in different pathways (Bevan, Nature, 325:192, 1987; Braciale, et al., Immunol. Rev., 98:95, 1987; Germain, Nature, 322:687, 1986):
(1) The MHC class I-related proteolytic system is present in virtually all cells for the purpose of degrading highly abnormal proteins and short-lived molecules or viral proteins. This proteolysis is thought to be non-lysosomal and to involve ATP-dependent covalent conjugation to the polypeptide ubiquitin (Goldberg, et al., Nature, 357:375, 1992). Peptide fragments, possibly in association with a larger proteasome complex, are then postulated to enter into the endoplasmic reticulum or some other type of exocytic compartment (other than the endocytic/lysosomal compartment). There they bind to MHC class I molecules and follow the constitutive secretary pathway from the endoplasmic reticulum through the Golgi to the cell surface where they are presented by the MHC I protein to the CD3-CD8 cytotoxic T cell antigen receptor.
(2) The MHC class II-related process by which foreign antigens are processed in antigen presenting cells (APC) cells is generally believed to occur in an endocytic pathway. Antigens taken into the cell by fluid-phase pinocytosis, absorptive endocytosis, or phagocytosis enter into a late endosomal/lysosomal compartment where large molecules are converted to peptides by digestion through proteases and other hydrolases. During this process, the immunodominant smaller peptides come in contact with and are bound by MHC class II molecules and the peptides are carried to the cell surface. On the cell surface of APC, these short peptides in conjunction with MHC class II molecules bind the CD3-CD4 complex on the surface of helper T cells, activating the replication and immune function of these cells. Following this interaction, helper T cells release lymphokines that stimulate the proliferation and differentiation of leukocytes and inhibit their emigration from the site of infection. In general, the activation of helper T cells by peptide-loaded APC is required for optimal B cell and T cell action, and thus is necessary for proper immune system function.
Some endogenous proteins may also enter the MHC class II system for antigen presentation (Malnati, et al., Nature, 357:702, 1992; Polydefkis, et al., 1990). It is postulated that endogenously-produced membrane antigen, which remains attached to the luminal/extracellular membrane by a hydrophobic anchor sequence, can recycle to the endosomal/lysosomal compartment by first reaching the surface of the cell via bulk flow followed by endocytic uptake and subsequent processing by the normal class II pathway for processing of exogenous antigens. MHC class II molecules may also present some antigenic determinants derived from endogenous proteins that are sequestered in the endoplasmic reticulum or other compartments and are then processed in salvage pathways to the lysosome (Brooks, et al., Proc. Nat'l. Acad. Sci. USA, 88:3290, 1991).
Other possible processing pathways for presentation of endogenously derived or cytosolic proteins to MHC class II-restricted T cells have also been described in some but not all experimental systems. These appear to be less efficient that the class I-associated process, and are not well understood (Moreno, et al., J. Immunol., 147:3306, 1991; Jaraquemada, et al., J. Exp. Med., 172:947, 1990). Alternative types of antigen presenting cells with different pathways for protein processing have been suggested, as well as the possibility of different proteases. The antigen-presenting capacity of cells bearing MHC class II shows variation according to cell type and is likely to be related to the proteolytic machinery and intracellular routes followed by antigen and MHC class II molecules (Peters, et al., Nature, 349:669, 1991).
The exact site of antigen processing and association of processed peptides with MHC class II in the endosomal/lysosomal pathway is as yet unclear. Data have been presented suggesting that MHC class II molecules meet with endocytosed proteins in the early endosomal compartment (Guagliardi, et al., Nature, 343:133, 1990). Partially processed antigens and easily degradable antigens may yield peptides that can combine with MHC class II in the early endosomal compartment. However, evidence is mounting that the major site of antigen processing and association with MHC class II occurs either in the late endosome, the lysosome, or a distinct compartment related to the lysosome (Neefjes, et al., Cell, 61:171, 1990). Recent studies describe a distinct vesicular compartment with lysosomal properties and characterized by high concentration of lysosomal-associated membrane protein (LAMP-1) and MHC class II molecules (Peters, et al., 1991).
The available data suggest the following sequence of events in the intracellular transport of MHC class II molecules: MHC class II molecules with the invariant chain are assembled in the endoplasmic reticulum and transported through the Golgi in common with other membrane proteins including MHC class I. The molecules are then targeted to specific endosomal/lysosomal organelles by an unknown mechanism, segregating from the MHC class I molecules which follow a constitutive route to the cell surface. In the endocytotic/lysosomal route, the invariant chain is removed from MHC class II by proteases acting in an acidic environment. At the same time, antigenic fragments of proteins that have entered the endocytic/lysosomal pathway are generated by these proteases and the resulting peptides bind to the class II molecules and are carried to the cell surface.
As described herein, the lysosomal/endosomal compartment is composed of membrane-bound acidic vacuoles containing LAMP molecules in the membrane, hydrolytic enzymes that function in antigen processing, and MHC class II molecules for antigen recognition and presentation. This compartment functions as a site for degradation of foreign materials internalized from the cell surface by any of a variety of mechanisms including endocytosis, phagocytosis and pinocytosis, and of intracellular material delivered to this compartment by specialized autolytic phenomena (de Duve, Eur. J. Biochem., 137:391, 1983).
The biosynthesis and vacuolar targeting mechanisms of the hydrolytic enzymes present in the lysosomal/endosomal compartment have been extensively studied (Kornfeld & Mellman, Ann. Rev. Cell Biol., 5:483, 1989). Newly synthesized hydrolases in the Golgi apparatus acquire mannose 6-phosphate groups that serve as specific recognition markers for the binding of these enzymes to mannose 6-phosphate receptors which are then targeted in some unknown manner to a prelysosomal vacuole. There the receptor-enzyme complex is dissociated by low pH, and the receptors recycle to the Golgi apparatus, while the enzyme-containing vacuole matures into a lysosome.
Studies of the structure and function of the lysosomal membrane were initiated in 1981 by August and colleagues with the discovery of major cellular glycoproteins that were subsequently termed lysosomal-associated membrane proteins one and two (LAMP-1 and LAMP-2) due to their predominant localization in the lysosomal membrane (Hughes, et al., J. Biol. Chem., 256:664, 1981; Chen, et al., J. Cell Biol., 101:85, 1985). Analogous proteins were subsequently identified in rat, chicken and human cells (Barriocanal, et al., J. Biol. Chem., 261:16755, 1986; Lewis, et al., J. Cell Biol., 100:1839, 1985; Fambourgh, et al., J. Cell Biol., 106:61, 1988; Mane, et al., Arch. Biochem. Biophys., 268:360, 1989). Typically, LAMP-1, as deduced from a cDNA clone (Chen, et al., J. Biol. Chem., 263:8754, 1988) consists of a polypeptide core of about 382 amino acids (Mr.congruent.42,000) with a large (346-residue) intraluminal amino-terminal domain followed by a 24-residue hydrophobic transmembrane region and short (12-residue) carboxyl-terminal cytoplasmic tail. The intraluminal domain is highly glycosylated, being substituted with about 20 asparagine linked complex-type oligosaccharides and consists of two .about.160-residue homology units that are separated by a proline/serine-rich region. Each of these homologous domains contains 4 uniformly spaced cysteine residues, disulfide bonded to form four 36-38-residue loops symmetrically placed within the two halves of the intraluminal domain (Arterburn, et al., J. Biol. Chem., 265:7419, 1990, see especially FIG. 6). The LAMP-2 molecule is highly similar to LAMP-1 in overall amino acid sequence (Cha, et al., J. Biol. Chem., 265:5008, 1990).
Another glycoprotein, described as CD63, MEA491 or LIMP 1, is also found in lysosomal membranes, as well as other in vacuolar structures (Azorza, et al., Blood, 78:280, 1991). This molecule is distinctly different from the LAMPs, with a core polypeptide of about 25,000 kDa and four transmembrane domains, but it has a cytoplasmic structure and sequence similar the LAMP molecules. There is also extensive amino acid sequence similarity between this protein and a family of other molecules that also contain four membrane spanning domains, including the Schistosoma mansoni membrane protein SM23, CD37, the tumor-associated antigen CO-029, and the target of an antiproliferative antibody-1.
Lysosomal acid phosphatase (LAP) is a hydrolytic enzyme that is also initially present in the lysosomal membrane, where it is subject to limited proteolysis that generates the soluble mature enzyme (Peters, et al., EMBO J., 9:3497, 1990). The protein has little sequence homology to the other described lysosomal membrane components, but it does contain a targeting sequence in the 19 residue cytoplasmic tail of the molecule (Pohlmann, et al., EMBO J., 7:2343, 1988).
LIMP II is an additional glycoprotein present in the membrane of lysosomes and secretory granules with lysosomal properties (Vega, et al., J. Biol. Chem., 266:16818, 1991). A sequence near the amino-terminus with properties of an uncleavable signal peptide and a hydrophobic amine acid segment near the carboxyl end suggest that the protein is anchored in cell membranes at two sites by two short cytoplasmic tails at the amine and carboxyl-terminal ends of the protein. The molecule does not have sequence hemology to any of the other described lysosomal membrane protein, but is highly similar to the cell surface protein CD36 which is involved in cell adhesion.
A number of other proteins have biological functions that also involve trafficking or targeting to or through vacuoles that may functionally involve the lysosomal/endosomal compartment. Examples of the most extensively characterized of these proteins at this time are as follows:
1. Cell Surface Receptors: PA2 2. Mannose 6-phosphate Receptor and Lysosomal Hydrolases: PA2 3. MHC Class II Molecule: PA2 4. Other Lysosomal/Endosomal Membrane Proteins:
Many cell surface receptors are known whose function is to bind and carry ligands into the cell. Examples include receptors for the low density lipoprotein (LDL, Chen, et al., J. Biol. Chem., 265:3116, 1990), insulin (Rajagopalam, et al., J. Biol. Chem., 266:23068, 1991), epidermal growth factor (Helin and Beguinot, J. Biol. Chem., 266:8363, 1991), polymeric immunoglobulin (Poly-Ig, Breitfield, et al., J. Biol. Chem., 265:13750, 1990), transferring (Collawn, et al., Cell, 63:1061, 1990), cation-dependent and independent mannose 6-phosphate receptors (MPR, Johnson, et al., Proc. Nat'l. Acad. Sci. USA, 87:10010, 1990; Canfield, et al., J. Cell Biol., 266:5682, 1990; Jadot, et al., J. Biol. Chem., 267:11069, 1992), and CD3 (Letourneur and Klausner, Cell, 69:1143, 1992). Trafficking of these receptors is commonly into an endosomal, and sometimes the lysosomal compartment. A well known mechanism includes the functional dissociation of the receptor-ligand complex in the acidic environment of the endosomal/lysosomal vacuole, releasing the ligand in the cell with the subsequent recycling of the receptor to the plasma membrane.
A highly characterized mechanism for delivering hydrolytic enzymes to lysosomes is the mannose 6-phosphate receptor which specifically recognizes the mannose 6-phosphate residues selectively added to these enzymes in their biosynthetic pathway (for review see Kornfeld and Mellman, 1989). This receptor targets the hydrolysases to a committed prelysosomal compartment where the membrane-bound receptor dissociates from the soluble hydrolase, and the receptor recycles to the Golgi or to the plasma membrane while the hydrolase-containing vacuole matures into or fuses with the lysosomal vesicle marked by the presence of the LAMP molecules.
The MHC class II molecule is also colocalized with the LAMP proteins in the endosomal/lysosomal compartment, where it binds to peptide fragments produced from molecules processed in this compartment by proteolytic enzymes. There is evidence that the targeting signal for this localization resides in the cytoplasmic tail of the invariant chain associated with the MHC class II molecule.
In additional to the proteins described above as components of the endosomal/lysosomal membrane, there is evidence for the presence of a number of other lysosomal/endosomal membrane proteins to serve a variety of functions associated with the structure or function of the vesicle, such as transport molecules, receptors or specific adhesion, association or signal molecules.
The localization of the lysosomal membrane glycoproteins is controlled by a targeting mechanism independent of the well defined mannose 6-phosphate receptor (MPR) pathway for hydrolytic lysosomal enzymes (Kornfeld and Mellman, 1989). Kinetic analysis of intracellular transport and targeting of newly synthesized LAMP-1 and other similar proteins indicate that the molecule is synthesized in the endoplasmic reticulum, processed in the Golgi cisternae and transported to lysosomes within one hour of its biosynthesis, without detectable accumulation in the plasma membrane (Barriocanal, et al., 1986; D'Sousa, et al., Arch. Biochem. Biophys., 249:522, 1986; Green, et al., J. Cell Biol., 105:1227, 1987).
The eleven amino-acid sequence of the cytoplasmic tail of LAMP-1 and other similar lysosomal membrane glycoproteins has the following sequence: Arg.sup.372 -Lys.sup.373 -Arg.sup.374 -Ser.sup.375 -His.sup.376 -Ala.sup.377 -Gly.sup.378 -Tyr.sup.379 -Gln.sup.380 -Thr.sup.381 -Ile.sup.382 -COOH (Chen, et al., 1988). Studies of the signals that target these proteins to lysosomes have focused on this sequence and it was shown that Tyr.sup.379 is critical for lysosomal targeting and that His.sup.376, Ala.sup.377, and Gly.sup.378 are unimportant in the targeting of the protein (Williams and Fukuda, et al., J. Cell Biol., 111:955, 1990).
A cytoplasmic Tyr is also critical for internalization from the cell surface of several receptors including low density lipoprotein (LDL) (Chen, et al., 1990), insulin (Rajagopalam, et al., 1991), epidermal growth factor (Helin and Beguinot, 1991), polymeric immunoglobulin (Poly-Ig) (Breitfield, et al., 1990), transferrin (Collawn, et al., 1990), cation-dependent and independent mannose 6-phosphate receptors (MPR) (Johnson, et al., 1990; Canfield, et al., 1990; Jadot, et al., 1992), and CD3 (Letourneur and Klausner, 1992). In the case of CD3, the molecule also utilizes a dileucine motif in the targeting mechanism.
Traditional vaccines rely on whole organisms, either pathogenic strains that have been killed or strains with attenuated pathogenicity. On the one hand, these vaccines run the risk of introducing the disease they are designed to prevent if the attenuation is insufficient or if enough organisms survive the killing step during vaccine preparation. On the other hand, such vaccines have reduced infectivity and are often insufficiently immunogenic, resulting in inadequate protection from the vaccination.
Recently, molecular biological techniques have been used in an attempt to develop new vaccines based on individual antigenic proteins from the pathogenic organisms. Conceptually, use of antigenic peptides rather than whole organisms would avoid pathogenicity while providing a vaccine containing the most immunogenic epitopes. However, it has been found that pure peptides or carbohydrates tend to be weak immunogens, seeming to require a chemical adjuvant in order to be properly processed and efficiently presented to the immune system. A vaccine dependent on T cell responses should contain as many T cell epitopes as would be needed to stimulate immunity in a target population of diverse MHC types. Further, since T cell recognition requires intracellular protein processing, vaccine preparations facilitating internalization and processing of antigen should generate a more effective immune response. Previous attempts to direct antigens to MHC molecules (see U.S. Pat. No. 4,400,276) were not effective because the antigen processing step was evaded. A successful hepatitis B vaccine has been prepared using cloned surface antigen of the hepatitis B virus, but this appears to be due to the tendency of the hepatitis surface antigen molecule to aggregate, forming regular particles that are highly immunogenic.
It is now well known that tumors express antigens that are capable of being recognized as foreign from host antigens by the T cell arm of the immune system and there are many potential types of tumor specific antigens:
In many cases, it has been demonstrated that peptides derived from altered genetic sequences of the sort described above can associate with either MHC class I or MHC class II molecules and be recognized by the appropriate helper or cytotoxic T cells.
The major thrust of cancer immunotherapy is the identification of these tumor specific antigens and then the development of immunization strategies that will most effectively generate T cell dependent immunity against these antigens. For example, studies indicate that vaccinia virus recombinant vaccines containing either the SV40 T antigen genes or the E6 and E7 genes from HPV or influenza nucleoprotein will protect animals against subsequent challenges with tumor cells that express these proteins as tumor antigens. The protection is associated with the generation of antigen specific responses among T cells in host.
Any strategy which would enhance the presentation of a particular antigen on MHC molecules of host antigen presenting cells would, in fact, enhance the immunization potential of such a viral based strategy for human cancer. The equivalent arguments can be made for generation of enhanced vaccine efficacy for viral infections such as HIV.